INTRODUCTION

The glycan composition of therapeutic glycoproteins may impact the biological activity, stability, pharmacokinetics, pharmacodynamics, efficacy, and immunogenicity of the molecule. The glycosylation pattern, or specific aspects of it such as the degree of sialylation, may form part of the product characterization and release specifications. One approach to ensuring the consistency of glycosylation and compliance of the product with specifications is to quantify specific monosaccharides present in the product. Monosaccharides are released by degradation of the product and quantified by chromatographic approaches. The most commonly determined sugars are N-acetylneuraminic acid (Neu5Ac) and its variant N-glycolylneuraminic acid (Neu5Gc). These monosaccharides can be selectively released from the protein either by dilute acid hydrolysis or by enzymatic methods, whereas increasingly aggressive hydrolysis conditions release neutral monosaccharides including mannose, galactose, and fucose, and then glucosamine and galactosamine. Separation and quantification of released monosaccharides by high-performance liquid chromatography (HPLC) can be achieved with or without fluorophore labeling.